Periyannan Rajeswari Prem Kumar, Soderberg Lovisa M, Yacoub Alia, Leijon Mikael, Andersson Svahn Helene, Joensson Haakan N
Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH Royal Institute of Technology, Tomtebodavägen 23A, 171 65 Solna, Sweden.
Department of Microbiology, National Veterinary Institute (SVA), Ulls väg 2B, SE 751 89 Uppsala, Sweden.
J Microbiol Methods. 2017 Aug;139:22-28. doi: 10.1016/j.mimet.2017.04.007. Epub 2017 Apr 20.
We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.
我们展示了一种使用荧光颜色编码的Luminex®微球检测多种病原体DNA生物标志物的液滴PCR工作流程。该策略能够利用具有数百种可区分荧光编码的市售微球集对多个单重液滴PCR进行编码。此工作流程提供了超越当前多重液滴PCR中常用的荧光检测探针（如TaqMan探针）所提供的有限数量的可扩展性。该工作流程针对与靶标特异性捕获寡核苷酸偶联的三种不同Luminex微球集进行了验证，以检测感染家禽的三种微生物的杂交情况：禽流感、传染性喉气管炎病毒和空肠弯曲菌。在该检测中，通过PCR在单分散皮升液滴中平行地用荧光标记引物扩增靶标DNA，以避免扩增偏差。Luminex检测微球的颜色编码允许对该检测中使用的不同微球集进行同时且准确的分类。杂交检测从每个液滴平均靶标浓度为单个DNA模板分子的样品中以高特异性检测到所有三种微生物的靶标DNA。此工作流程证明了利用颜色编码的Luminex检测微球提供的可扩展性增加液滴PCR检测面板以并行检测大量靶标的可能性。
